Amino Acid Alphabet

8/7/2019 Amino Acid Alphabet

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Amino acid alphabet

Twenty amino acids are used in all proteins with the exception of:

selenocysteine: occurs in enzymes from all three domains (archae, eubacteria,

eukaryotes) and is coded by the amber stop codon (UGA)

Synthesized in situ from Ser-tRNA (no aminoacyl transferase for selenocysteine)

pyrrolysine: occurs in some methanogenic archaea in enzymes as part of theirmethane-producing metabolism

selenium

instead

of sulfur


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The relative abundances of individual amino acids on primordial earth

were probably different than they are today and thus the amino acid

alphabet may have consisted of fewer (or more) than 20 amino acids

Osawa et al Microbiol Review 56, 229 (1992)

How did the current amino acid alphabet evolve?

The standard alphabet may consist of the smallest and cheapest amino acids that

form a functional protein libraryminimalist thinking

Protein structures are often tolerant to amino acid substitutions

Helps identify homologous proteins and is crucial for homology modeling

The sequence structure relationship may become more apparent if

proteins can be engineered using fewer than 20 amino acids


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T4 lysozyme with A40-49

Can we engineer functional protein with well defined structure using

fewer than 20 amino acids and what is the minimum number of aminoacids required for the task?

Mutate multiple residues simultaneously to see if a large number of

amino acids can be altered simultaneously to a much smaller set without

perturbing the structure and function irreversibly

Residues 39 to 50 T4 lysozyme form an alpha helix

mutate residues 40 to 49 with 10 consecutive Ala

39-LNAAKSELDKAI-5039-LAAAAAAAAAAI-50

Heinz et al. PNAS 89, 3751 (1992)


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Depending on the number of substitutions, the mutants are destabilized

by Tm = - 8.5 10.7 C and G = 2.1 2.5 kcal/mol at pH 3.0 and

6.7

Yet they preserve the overall the structure

main chain atoms overlap


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Minimal homeodomain

Homeodomain is a conserved DNA binding domain that

is ubiquitous among developmental regulatory proteins

from nematodes to humans

Consists of 3 alpha helices and a long N-terminal arm

A consensus sequence can be generated by sequencecomparison and identify residues that are important for

tertiary structure and DNA binding activity

Shang et al, PNAS 91, 8373 (1994)

Homeodomain proteins also exhibit sequence specificity contributed by

nonconserved residues


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Goal: Design a minimal

homeodomain containing all

consensus residues, in which all

non-conserved residues have beensubstituted with alanine

Consensus residues contain

sufficient sequence information to

construct the scaffold for specificDNA recognition

Non-conserved residues in the N-

terminal arm are required for high

affinity DNA binding as well as fortertiary structure

It is possible to design a vastly

simplified high affinity mutant with a

large number of Alagel shift assay


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Gel shift assay

Gel retardation assay

EMSA : electrophoretic mobility shift assay

PAGE : polyacrylamide gel electrophoresis

movem

ent


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Peptitergent to solubilize membrane protein

Structural determinants of alpha helical peptides are well understood

Alpha helical peptide of 24 residue peptide-detergent PD1

(peptitergent) was designed to self-associate and to solubilize

transmembrane proteins

Schafmeister et al, Science 262, 734 (1993)

PD1 is amphipathic with Ala and Leu on one side


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Four helix bundles are versatile. Design and characterize an antiparallel

four helix bundle DHP1 using an alphabet of seven amino acids

Interhelical loops consist of 3, 4 and 3 glycines

Lys, Glu, Gln, Ala, Leu, Gly, Ace (capping residue)

Helices cross each other at -20

Hydrogen/deuterium exchange shows that amideprotons are shielded from solvent exchange

with a protection factor > 1.6 x 10^4

Cf. For molten globule proteins, the protection

factors are < 100

In native proteins, between 1000 5 x 10^8

Sufficiently ordered structure can be designed

using only seven amino acids

Schafmeister et al, NSB 4, 1039 (1997)

Helix bundle with 7 amino acids


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Hydrogen-deuterium (H/D) exchange

Amide protons constantly exchange with the solvent

If the protein is submerged in heavy water (D2O instead of H2O), then after

losing its original hydrogen an amide nitrogen will pick up a deuterium

The rate of exchange can be monitored by mass spec or by NMR

H/D exchange can be used to :

Measure the rate of folding and unfolding

Solvent accessibility at protein-protein interfaces

Determine disordered regions of native, folded proteinhence stability as

well

green: no exchange

blue: too fast

red: just right


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Binary patterning

Is it possible to design a protein by specifying just its hydrophobicity pattern without

the amino acid identities?

The hydrophobicity pattern on a helix repeats itself roughly every 3.6 amino acids.

Test if a randomized peptide with the hydrophobicity pattern of a helix actually folds

to a helical protein

Kamtekar et al,

Science 262, 1680(1993)


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Binary patterned library

Identify the residue positions required to be hydrophobic and hydrophilic

Synthesize genes to introduce F, L, I, M or V at 24 hydrophobicpositions, and D, E, K, N, Q, and H at 32 hydrophilic positions

total diversity: 5 ^ 24 + 6 ^ 32 = 4.7 x 10^41

Heptad repeat


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ligand binding site

Beta sheet protein with reduced alphabet

Helices and helical domains are in general easier than beta sheets

Can a beta sheet protein be similarly engineered using a reducedalphabet?

SH3 domain is a small beta barrel like structure

that binds a proline-rich peptide

Replace all residues not involved in ligand binding

with a small set of amino acids including

I, K, E, A, G

Display mutants on phage for selection (more on

this later in the semester)

Beta sheet protein can be reconstructed using a simplified alphabet

Riddle et al, NSB 4, 805 (1997)


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Enzyme from 9 amino acids

Can a reduced amino acid alphabet support enzymatic catalysis?

Designing/engineering an enzyme is difficult because of the precise

positioning of residues required for catalysis

Chorismate mutase (CM) catalyzes the rearrangement of chorismate to

prephenate and is essential for the biosynthesis of aromatic residues

The enzyme is a domain-swapped homodimer of

three helices and two loops

Assay: express a mutant CM in a strain deficient in the

enzyme (auxotrophic) and select for viable clones

Walter et al, JBC 280, 37742 (2005)


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1. Active site: Q88, R11, R28, R51

E > N > D

kcat is 3-fold lower than wild type

kM is 40 fold higher

i.e. second order rate constant ~ 1/120

1. Loop #2: H66, V68 to polar and nonpolar

four active mutants out of 48

1. Loop #1: G43-I44-P45-I46D, E, N, K, R, F, I, L, M

five active mutants out of 58,564

i. + ii. mutantiii. wild type

iv. pos control

v. neg control

Amino Acid Alphabet

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